Nuclear biochemical changes that are characteristic of apoptosis and can be induced by a variety of cytotoxic stimuli are traditionally associated with the executive or late stages of this form of cell death. The long-term objective of this proposal is to advance our understanding of nuclear participation in the initiative or early stages of apoptosis. The main hypothesis to be studied is that DNA-damaging agents stimulate apoptosis by imparting changes at the nuclear level, resulting in the release of factors into the cytosol, which target mitochondria and stimulate the release of cytochrome c. Because of the apparent ability of Hsp27 to exert its anti-apoptotic effect against a variety of stimuli, including DNA-damaging agents, at the level of cytochrome c release, the mechanism regulating the cytoprotective potential of this protein is a complementary focus of this proposal. The specific aims are: 1) To characterize the ability of nuclei to stimulate established mitochondrial changes typical of apoptosis in response to DNA-damaging agents and the ability of Bcl-2/Bcl-XL to mitigate this effect; 2) To determine the mechanism(s) regulating the ability of Hsp27 to prevent apoptosomal caspase activation by blocking cytochrome c release. Completion of these studies will help clarify the ability of nuclear events, induced by DNA damage, to evoke mitochondrial changes characteristic of apoptosis and the mechanism responsible for Hsp27-mediated cytoprotection at the mitochondrial level.